This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Cryo-electron microscopy (cryo-EM) combined with three-dimensional image reconstruction techniques and newly developed time-resolved methods provide a powerful way to examine virus-host cell interactions as well as the dynamic structural changes that occur during viral cell entry. In fact these methods often represent the only feasible structural approach for large (100 million Dalton) macromolecular complexes and virus/receptor complexes with highly mobile receptor binding sites, as has been postulated for adenovirus. These proposed studies will elucidate the structural events during adenovirus/receptor binding at thle cell surface, intemalization into coatedpits, and escape ofthe virus particle from the endosome.
Specific aims i nclude imaging the adenovirus particle complexed with a Fab fragment of a monoclonal antibody that blocks cell entry in order to localize the Arg-Gly-Asp integrin binding site on the penton base viral component. In addition, the structure of the virus complex ed with a soluble form of the internalization receptor, the integrins avb3 and avb5, will be solved in order to evaluate possible structural changes that occur upon receptor binding. We will also examine conformational changes in the virus and virus/receptor complexes as a consequence of varying the pH value to mimic both the extracellular (pH 8) and early endosomal (pH 6.0 - 6.5) environments. Rapid freezing (within milliseconds) after low pH treatment allows the trapping of structural intermediates. A further objective involves calculating an image reconstruction of the adenovirus temperature sensitive mutant, tsl, which is defective for cell entry. The structural differences between the tsl mutant and wild type will be analyzed to isolate elements essential for productive adenoviral cell entry. These cryo-EM structural studies will provide a greater understanding of the cell entry process of adenovirus, a promising vector for in vivo gene therapy.
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